Lipoprotein lipase (LPL) is required for uptake of plasma lipoprotein triacylglycerol (TG) by tissues. It is synthesized in parenchymal cells and transferred and attached by proteoheparan sulfate to capillaries, where it acts. Active LPL is a noncovalent homodimer of N-linked glycosylated subunits. Active dimeric LPL has higher affinity for heparin than inactive monomeric LPL. Sucrose gradient centrifugation and heparin-Sepharose chromatography showed that most LPL dimers in normal brown adipocytes were active and had high heparin-affinity (HA). When Golgi mannosidase I was blocked with DMM (1-deoxymannojirimycin), only 60% of the dimers were active and had high HA, and the rest were inactive and had low HA. BFA (brefeldin A), which blocked LPL transport from ER (endoplasmic reticulum) and relocated Golgi components to ER, increased to 100% the proportion of LPL dimers in DMM cells that were active and had high HA. Blockade of glucosidase I activity in ER with CST (castanospermine) resulted in synthesis of inactive LPL which had low HA and was retained in ER primarily as higher oligomers. CST cells treated with BFA contained LPL dimers, two-thirds of which were active and had high HA. The findings show that LPL can be present in two forms of dimers in cells. That LPL maturation occurred in ER of cells in which Golgi enzymes were relocated to ER by BFA suggests that processing by Golgi components is necessary for dimerization, high HA and activity of LPL. Our findings suggest that LPL subunits in active dimers are aligned so that heparin binding sites are on the back side and active site regions on the front side of the dimer, allowing active site lids to open freely upon interfacial activation, and both active site regions to interact with the surface of TG-rich lipoproteins. Combined lipase deficient (cld/cld) mice have functional deficiencies of both LPL and hepatic lipase. The adrenal glands of cld/cld mice are normal in size, but they have no hepatic lipase activity (required for uptake of plasma CE by adrenals) and 93% less cholesteryl ester (CE) than normal. Plasma concentration of HDL-CE, the primary source of cholesterol for adrenals, is also very low (10% normal) in cld/cld mice. These findings indicate that cld/cld mice probably die of adrenal insufficiency, secondary to impaired delivery of cholesterol to the adrenals.